Abstract
The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by HR are very low. In this study, we compared two methods that have been reported to enhance frequencies of HR in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting (IPGT) approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of HR events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by HR at frequencies of 0.12% using the IPGT approach, while both approaches using viral replicons did not yield any trichome-bearing plants.
Highlights
Gene targeting (GT) means integration of foreign DNA into a cell’s genome by homologous recombination (HR) (Paszkowski et al, 1988)
We used a CRISPR/Cas9 approach to generate a T-DNA free glabrous Arabidopsis mutant that contains a deletion of 10 bp in the master regulator gene for trichome formation, GL1
Since the mutant line was created by CRISPR/Cas9, a protospacer adjacent motif (PAM) sequence was already present at the site of mutation and the predicted Cas9 cleavage site (3 bp upstream of the PAM sequence) matched the desired integration site for the missing 10 bp (Supplementary Figure S1)
Summary
Gene targeting (GT) means integration of foreign DNA into a cell’s genome by homologous recombination (HR) (Paszkowski et al, 1988). GT has been a long-term goal for plant scientists since it allows modifying an endogenous gene in planta or integrating a transgene at a specific position in the genome (Puchta, 2002). GT is achieved in lower eukaryotes, such as yeast, or the moss Physcomitrella patens, because they include foreign DNA predominantly through highly efficient HR pathways. GT is not achieved in higher plants, since they integrate foreign DNA via illegitimate recombination through the non-homologous end joining (NHEJ) pathway (Puchta, 2005). GT frequencies in higher plants can be increased by two orders of magnitude by inducing a double strand break (DSB) in the target locus with sequence specific nuclease (SSN) (Puchta et al, 1996). The most recent addition to the SSN has been the CRISPR/Cas system (Cas system)
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